All coordinated movements such as locomotion, breathing, and swallowing, depend on the rapid transfer of information between motor neurons and the skeletal muscle fibers they innervate that takes place at a specialized contact called the neuromuscular junction. Disruption of this system by disease, as occurs in various myasthenic disorders, spinal muscular atrophy, and amyotrophic lateral sclerosis, toxins such as botulinum toxin, or traumatic injury, results in muscle weakness and even death. Thus, understanding the mechanisms responsible for the formation and maintenance of the neuromuscular junction has the potential for significant public health benefits by identifying new therapeutic targets for a wide range of disorders that impact the function of this important synapse. A major success in the field has been the elucidation of the role of a protein called agrin in mediating the motor neuron-induced differentiation of the postsynaptic apparatus formed by the muscle fiber. Agrin exerts its effects through a muscle fiber membrane receptor complex consisting of LRP4, a low-density lipoprotein receptor-related protein, and MuSK, a muscle-specific tyrosine kinase. Curiously, however, not all forms of agrin at the neuromuscular junction are competent to activate the LRP4/MuSK receptor and several studies have suggested these "inactive" agrin species play a role regulating growth and differentiation of motor axons and axon terminals. The receptor that might mediate these presynaptic effects is unknown but a good candidate is the a3 Na,K-ATPase (NKA), a neuron-specific isoform of the sodium-potassium pump that binds all forms of agrin. Agrin-dependent regulation of the a3 pump modulates excitability of central nervous system neurons, as well as the levels of cytoplasmic calcium and other second messengers implicated in regulating synaptic function, growth and maturation. The a3 NKA is also present on motor axon terminals, raising the possibility that the agrin/a3 NKA pathway plays a related role at the neuromuscular junction. To test this hypothesis we will examine the effect of knocking out the a3 NKA on development of the neuromuscular junction, the role of the a3 NKA in regulating growth of motor axons and dendrites, and the role of agrin/a3 NKA in modulating neuromuscular synaptic transmission. Previous studies of agrin function at the neuromuscular junction have focused on its interaction with the postsynaptic LRP4/MuSK receptor. This would be the first to examine the significance of an alternate agrin signaling pathway at this important synapse. Neuromuscular diseases such as the myasthenic disorders, spinal muscular atrophy, amyotrophic lateral sclerosis are characterized by profound muscle weakness linked to a decline in neuromuscular synaptic transmission. We believe the agrin/a3 NKA pathway will prove to be a novel therapeutic target that can be exploited for the treatment of these and related neuromuscular disorders. PUBLIC HEALTH RELEVANCE: The neuromuscular junction is the vital connection between the nerves and muscle fibers responsible for respiratory and voluntary movements. In many neuromuscular diseases, such as spinal muscular atrophy, myasthenia gravis and congenital myasthenic syndrome, the function of the neuromuscular junction is compromised resulting in profound muscle weakness and death. Here we examine a novel mechanism whereby agrin, a protein known for its role in directing the formation of the post-synaptic apparatus of the neuromuscular junction, influences growth and function of the pre-synaptic motor axon terminal. This novel signal pathway represents a new therapeutic target for the treatment of neuromuscular disease.